This proposal addresses the mechanism by which the MCM2-7 helicase unwinds DNA at eukaryotic replication forks, a crucial, unsolved problem in the field of chromosome duplication. Prokaryotic replicative DNA helicases generally function as a single hexamer that translocates along one strand and excludes the other strand. However, the MCM2-7 complex may not fit this simple paradigm. In the G1 phase of the cell cycle, MCM2-7 is recruited into pre-Replication Complexes (pre-RCs) as numerous inactive dimers that encircle double-stranded DNA. At the G1/S transition, two protein kinases (CDK and DDK) cooperate with a large number of additional factors to activate the MCM2-7 helicase in a reaction whose mechanism is currently mysterious. Only a subset of MCM2-7 complexes is normally activated, raising the question of what happens to the other MCMs that are clamped tightly around dsDNA. To study the properties of the MCM2-7 complex in the context of vertebrate DNA replication, the applicant's laboratory employs a soluble cell-free system derived from Xenopus laevis eggs. A unique feature of this system is that it supports efficient replication of model DNA templates such as plasmids and ; phage DNA, both of which can be readily modified in specific ways. We will combine this cell-free system with DNA nano-manipulation and real-time single molecule imaging to address the following questions. First, using stretched, doubly- tethered lambda DNA as a substrate, we will determine how many MCM2-7 complexes are present in each pre-RC and whether unactivated MCMs are removed or mobilized when struck by a moving DNA replication fork. Second, we will collide the replisome with strand-specific DNA roadblocks to address whether MCM2-7 translocates along ssDNA or dsDNA during DNA unwinding. Finally, we will use single-molecule imaging approaches to probe the dynamic properties of the MCM2-7 helicase complex. Of particular interest is whether the rate of MCM2-7-mediated DNA unwinding slows down when the helicase uncouples from the replisome during replication stress. Together, our experiments will elucidate the molecular mechanism and dynamics of the MCM2-7 helicase and thereby greatly deepen our knowledge of how cells copy and maintain their genomes. )